Inhibition of 11-hydroxysteroid dehydrogenase type 2 as a potential
treatment for hyponatremia
[Author information removed by MSC]
XXX. Patients with loss-of-function mutations in 11β-HSD2 (11β-hydroxysteroid
dehydrogenase type 2) are characterized by sodium retention. The direct inhibition of this
enzyme could therefore represent a potential therapy for chronic hyponatremia, a condition
that is prevalent among elderly patients. To identify agents with the ability to bind and
modulate 11β-HSD2 specifically, a virtual screen of several compound libraries was
performed using a newly developed algorithm for detecting compounds with the required
physicochemical and structural characteristics. The top X candidate compounds identified by
the virtual screen were evaluated for their ability to interact with recombinant human 11βHSD2. One compound, DE2300c5, strongly bound to 11β-HSD2 without affecting either
11β-HSD1 or 17β-HSD2. Binding was confirmed in vitro using HEK293 cells, and
administration of DE2300c5 increased intracellular sodium levels in renal cortical cells. miR401 was unchanged by DE2300c5 administration. The findings of the present study suggest
that DE2300c5 may be a potential treatment for hyponatremia, which is a significant health
burden, and other electrolyte imbalances.
INTRODUCTION
XXX. The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) catalyzes the
conversion of the biologically active steroid hormone cortisol to its inactive form, cortisone
(Fig. 1). This reaction is co-factor dependent and relies on NAD+2. Cortisol binds to the MR
as tightly as its other natural ligand, aldosterone. In vivo, 11β-HSD2 is co-localized in tissues
where expression of the MR is high; the main role the enzyme in this setting is to regulate the
local concentration of cortisol and thereby prevent excessive binding to the MR. Activation of
the MR results in re-absorption of sodium ions, excretion of potassium ions and an associated
increase in blood pressure. The MR is a member of the nuclear receptor family, and it
transcriptionally regulates the alpha subunit of the epithelial Na(+) channel as well as other
ion transport machinery.
XXX
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For illustrative purposes only.
Dose-Response Curves
The 11β-HSD2 inhibition assays were performed at room temperature (25°C) in 35 mM Tris
buffer, pH 8.0, containing 20 mM NaCl. The total reaction volume in each well of the plate
was 100 μl and contained the following: 30 μl of each inhibitor (see Supplementary
Information), 30 μl of 10 mM NADH, 30 μl of 4 mM cortisol and 60 μl of 160 μg/ml 11βhydroxysteroid dehydrogenase-2. The reaction mixture was incubated at 25°C and then the
substrate was added. The product was transferred into the wells before fluorescence was
measured using a fluorometer. Each reaction was performed in triplicate using the inhibitors
at different concentrations (see Supplementary Information). A master mix was used to
initiate the reaction, and blanks for all inhibitors were included as controls. The pH of each
reaction was determined.
Crystallographic analysis
Crystallization trials were performed with bound DE2300c5 to investigate the mechanism of
ligand binding. Human recombinant 11β-HSD2 was overexpressed in the BL21 strain of E.
coli, and the purified extract was determined to be 98% pure using SDS-PAGE gels and a
densitometric analysis. The Bradford protein assay was used to determine the concentration of
the purified extract. The enzyme was then concentrated in 500 μM NADH buffer containing
1% glycerol, 1 mM EDTA, 50 mM Tris, pH 8, and 100 mM NaCl. The enzyme was then
assayed for activity. Finally, the sample was centrifuged at 2200 rpm for 5 to 10 minutes at
5°C to remove dust, precipitated protein, etc. Human recombinant 11β-hydroxysteroid
dehydrogenase-2 was crystallized at a final concentration of 32 mg/ml in pH 7.8 crystal buffer
(1% glycerol, 30 mM Tris, 45 mM NaCl, 5 mM EDTA).
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For illustrative purposes only.
Figure 3. The inhibition of 11β-HSD2 from each potential inhibitor in transfected cells.
DE2300c5 is compound 8. The experiments were performed using HEK293 cells.
Figure 4. The relationship between counts per minute (as detected by scintillation counting)
and inhibitor concentration for 11β-HSD2-transfected HEK293 cells. A dose-response plot is
shown for each candidate 11β-HSD2 inhibitor. The chemical structure and the IC50 are also
shown. The maximum line corresponds to substrate binding in the absence of a competitor.
DE2300c5 is compound 8. Error bars = S.E., N = 2.
Table 1. A summary of the results from the scintillation proximity assay (SPA) for six
selected compounds. Please see the Supplementary Information for further details.
Compound number
IC50 cell SPA
Calculated K i
Calculated Ki of SPA
of SPA (cell)
(recombinant)
3
10.4 ± 1.1 μM
1.2 μM
N/A
5
1.8 ± 4.04 μM
2.5 μM
N/A
DE2300c5
1.82 ± 0.03 nM
15 nM
1.7 nM ± 0.3
9
10.11 ± 8.1 μM
2.8 μM
N/A
10
9.61 ± 12.4 μM
5.7 μM
N/A
21
6.73 ± 2.06 μM
2.2 μM
N/A
Table 2. Summary information relating to the top compounds inhibiting 11β-HSD2 and the
query molecules.
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For illustrative purposes only.